Dairy cows are significant investments for dairy farmers, yet infertility is a major cause of dairy cow culling and economic loss. Enormous efforts, such as animal breeding and artificial insemination, have been and continue to be invested in ensuring improved breeding programs. The decline in reproductive performance in high-producing dairy cows is a major concern of farmers worldwide (Royal et al., 2000; Dobson et al., 2008). Major factors contributing to this poor performance in dairy cattle are low fertilization rate and early embryonic loss (Santos et al., 2004; Morris and Diskin, 2008). Although genetics account for about one-third of the decline in pregnancy rate of dairy cows (Shook, 2006), the identification of major genes affecting cow fertility has been challenging, probably due to the low accuracy of fertility data collected in the field and to the low heritability of this trait. The heritability of open days and pregnancy rate is about 0.04 (VanRaden et al., 2004).
Typically, artificial insemination in dairy cattle is successful only 30-35% of the time. The reasons for this are not clear. However, it is understood that both biological and environmental factors affect fertility rate. Some environmental factors such as heat, lack of precipitation, and other factors can cause stress in cattle and can drop the fertility rate to 10-15%. Commercial artificial insemination operations often shut down in July and August due to the drop in fertility caused by the hot, dry weather. It is also known that certain bulls are more fertile than others due to their genetic makeup. Identifying highly fertile bulls, however, is a time-consuming and expensive process. It can take 5-10 years of tracking the attempts of artificial insemination using semen from a bull before it can be certified as a quality bull.
Marker-assisted selection, on the other hand, can lower the high cost and reduce the extended time commitment of progeny testing currently used to improve sires, since young bull progeny could be evaluated immediately after birth or even prior to birth for the presence/absence of the marker, and young bulls that are determined by genetic testing to have undesirable markers would never be progeny tested.
There is thus a need for a method of genetically evaluating the bulls, as well as the cows, e.g., by genetic testing, to enable a quick and accurate evaluation of its fertility as well as the survival rate of embryos conceived therefrom.
Signal transducer and activator of transcription (STAT) proteins are a family of 7 structurally and functionally related proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 (Darnell, 1997). The STAT proteins are transcription factors that play important roles in cytokine signaling pathways (Kisseleva et al., 2002). Following their phosphorylation by janus-kinases (JAKs), STATs translocate to the nucleus to regulate transcription of different genes. The JAK/STAT pathway was found to be conserved in vertebrates (Hombría and Brown, 2002). Recent studies have shown that STAT proteins are involved in the fertilization process and in early embryonic development (Maj and Chelmonska-Soyta, 2007). Teglund et al. (1998) showed that disruption of the Stat5 gene leads to infertility in female mice as a result of small-sized or absent corpora lutea. Truchet et al. (2004) reported that Stat1 and Stat3 are expressed in mouse oocytes and preimplanation embryos and concluded that these 2 genes might have functional importance in early embryonic development because of their roles in the cell cycle and apoptosis. Takeda et al. (1997) reported that Stat3-deficient mice die before embryonic day 8.5 and concluded that Stat3 is an essential gene for early embryonic survival and that its deficiency cannot be compensated for by other STAT proteins. Khatib et al. (2008a, 2009) showed that the CC genotype in exon 8 of bovine STAT5A was associated with high fertilization and early embryonic survival rates.
Given that several genes of the JAK/STAT pathway have been found to be associated with fertility traits in cattle, STAT1 and STAT3—also members of this pathway—were chosen as candidate genes for fertilization rate and early embryonic survival in cattle. Previously, the present inventor has disclosed that single nucleotide polymorphisms (SNPs) in the STAT5A gene are associated with both milk production and fertility (U.S. patent application Ser. No. 12/267,076), and a SNP in the coding region of STAT1 gene is associated with increased milk yield, milk fat and protein percentages (U.S. patent application Ser. No. 11/624,053).
Interestingly, after their phosphorylation in the cytoplasm by the JAKs, STAT1 and STAT3 interact with each other by forming a heterodimer complex which translocates to the nucleus and binds specific DNA sequences (Kodama et al., 1997).
In order to overcome these challenges, the present inventor has constructed an in-vitro fertilization (IVF) system which has the advantages of a unified environment and well-isolated components of the embryonic development process. Indeed, using this system, SNPs in several genes and interactions between them have been found to be associated with fertilization and early embryonic survival rates (Khatib et al., 2008a,b; Khatib et al., 2009). There remains, however, a need to determine the single gene effects of STAT1 and STAT3 polymorphisms and their interactions on fertilization and embryonic survival rates.